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Pseudomonas putida CSV86: A Candidate Genome forGenetic BioaugmentationVasundhara Paliwal1, Sajan C. Raju2, Arnab Modak3, Prashant S. Phale3, Hemant J. Purohit1*
1 Environmental Genomics Division, CSIR-National Environmental Engineering Research Institute, Nagpur, India, 2 MEM-Group, Department of Biosciences, University of
Helsinki, Helsinki, Finland, 3 Department of Biosciences and Bioengineering, Indian Institute of Technology-Bombay, Powai, Mumbai, India
Abstract
Pseudomonas putida CSV86, a plasmid-free strain possessing capability to transfer the naphthalene degradation property,has been explored for its metabolic diversity through genome sequencing. The analysis of draft genome sequence of CSV86(6.4 Mb) revealed the presence of genes involved in the degradation of naphthalene, salicylate, benzoate, benzylalcohol, p-hydroxybenzoate, phenylacetate and p-hydroxyphenylacetate on the chromosome thus ensuring the stability of thecatabolic potential. Moreover, genes involved in the metabolism of phenylpropanoid and homogentisate, as well as heavymetal resistance, were additionally identified. Ability to grow on vanillin, veratraldehyde and ferulic acid, detection ofinducible homogentisate dioxygenase and growth on aromatic compounds in the presence of heavy metals like copper,cadmium, cobalt and arsenic confirm in silico observations reflecting the metabolic versatility. In silico analysis revealed thearrangement of genes in the order: tRNAGly, integrase followed by nah operon, supporting earlier hypothesis of existence ofa genomic island (GI) for naphthalene degradation. Deciphering the genomic architecture of CSV86 for aromaticdegradation pathways and identification of elements responsible for horizontal gene transfer (HGT) suggests that geneticbioaugmentation strategies could be planned using CSV86 for effective bioremediation.
Citation: Paliwal V, Raju SC, Modak A, Phale PS, Purohit HJ (2014) Pseudomonas putida CSV86: A Candidate Genome for Genetic Bioaugmentation. PLoS ONE 9(1):e84000. doi:10.1371/journal.pone.0084000
Editor: John R. Battista, Louisiana State University and A & M College, United States of America
Received August 27, 2013; Accepted November 11, 2013; Published January 24, 2014
Copyright: � 2014 Paliwal et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors are grateful to the Council of Scientific and Industrial Research (CSIR) for their facilities. The study has been supported through a grantfrom the Council of Scientific & Industrial Research, project number ESC0108 (Supra- Institutional Network Project). PSP thanks DST, Government of India, for aresearch grant. AM also thanks the CSIR, Government of India, for SRF. The funders had no role in study design, data collection and analysis, decision to publish, orpreparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: hemantdrd@hotmail.com
Introduction
Pseudomonas exhibits diverse metabolic capacities; which allow it
to survive in different ecological niches, including sites contam-
inated with pollutants such as aromatic compounds. The required
metabolic attributes are reflected by its large size genome
(generally .6 Mb). Pseudomonads have been reported for their
ability to exchange genetic information through horizontal gene
transfer (HGT) via phages, plasmids, transposons and genomic
islands (GIs), thus aiding in dissemination as well as evolution of
new diversified metabolic pathways [1]. These processes allow
sustained survival of genetic resources. Of these mobile genetic
elements (MGEs), GIs have especially been reported to code for
genes which render metabolic versatility, pathogenicity and heavy
metal resistance to microbes [2,3]. These capacities could be
exploited through genetic bioaugmentation for in situ breeding of
native population which not only ensures the survival of novel
genetic determinants, but also helps in enhancing the bioremedia-
tion process.
Pseudomonas putida CSV86 (hereafter referred to as CSV86), a
soil isolate, utilizes aromatic compounds like naphthalene, 1- and
2-methylnaphthalene, phenylacetic acid (PA) and p-hydroxyphe-
nylacetic acid (4-HPA), salicylate, benzylalcohol, benzoate and p-
hydroxybenzoate as the carbon source [4,5,6,7]. Strain CSV86
showed a novel property of preferential utilization of aromatic
compounds over glucose and co-metabolism of aromatics and
organic acids [8,9,10,11]. Though CSV86 lacks plasmid, the
naphthalene degradation property could be transferred by
conjugation which was found to be integrated in to the
chromosome of the transconjugants [12].
In the present study, the reported pathways for the utilization of
aromatic compounds have been annotated using the draft genome
sequence of CSV86 (6.4 Mb) [13]. Genome analysis revealed
additional catabolic pathways for aromatic compounds as well as
heavy metal resistance. These observations were further validated
by phenotypic (cell-growth and enzyme activity) experiments.
Based on these analyses, bioremediation and bio-augmentation
strategies can be developed for the effective remediation of
ecosystems polluted with aromatic compounds.
Materials and Methods
CSV86 draft genome assembly, ordering and annotationThe genome of Pseudomonas putida CSV86 was sequenced using
Roche 454 GS (FLX Titanium) platform. The 867,565 high
quality reads were assembled into 228 contigs with Newbler
Ver2.0, 454 assembly tool with sequence coverage of 61.08 fold
and average read length of 428 bp. Ordering of contigs was
performed using a tool, Mauve Contig Mover (MCM) [14]
available in Mauve software (http://gel.ahabs.wisc.edu/mauve.)
using P. putida S16 complete genome (NC_015733) as the
PLOS ONE | www.plosone.org 1 January 2014 | Volume 9 | Issue 1 | e84000
reference. P. putida S16 was also used as a reference for contig
scaffolding by using SIS program [15].
The genome was annotated using Rapid Annotations using
Subsystems Technology (RAST) v4.0 [16] and NCBI PGAAP
(Prokaryotic Genomes Automatic Annotation Pipeline) (http://
www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html). In NCBI
PGAAP, the 228 contigs were trimmed down to 209 due to quality
check and preprocessing of sequences. These contig were later
processed for annotation. The annotation by both RAST and
NCBI PGAAP tool was used to describe the genome of CSV86 in
this paper.
This Whole Genome Shotgun project has been deposited at
DDBJ/EMBL/GenBank under the accession no. AMWJ00000000.
The version described in this paper is the first version,
AMWJ01000000.
Comparative genomics and phylogenetic relationshipTaxonomic relationship. Phylogenetic relationship of
CSV86 was established using 16S rRNA gene sequences of 38
completely sequenced Pseudomonas species from NCBI database.
The alignment was carried out using ClustalW and the
phylogenetic tree was constructed using the maximum likelihood
algorithm (Hasegawa-Kishino-Yano model) with MEGA 5.2 [17].
In addition, MEGA 5.2 was also used for alignment and
constructing phylogenetic tree using promoter sequences of
naphthalene degradation genes.
Sequence variation in metabolic genes. Primary DNA and
protein sequences of CSV86 were compared with other closely
related species for similarity in catabolic pathways using NCBI blast
tools such as megaBLAST and BLASTp, respectively. Genes
involved in the degradation of aromatic compounds in CSV86 were
identified using RAST and NCBI PGAAP annotation along with
available literature, KEGG [18] and Metacyc [19] databases.
Comparative genome analysis using BRIG and
Mauve. BRIG (BLAST Ring Image Generator) [20] software
was used for the circular representation of multiple genome
comparison. The draft genome of CSV86 was used as the
reference genome and was compared with genome of P. putida S16
(NC_015733), P. putida KT2440 (NC_002947), P. entomophila L48
(NC_008027) and P. stutzeri CCUG 29243 (NC_018028).
Progressive alignment function of Mauve software with default
settings was used to compare the homology among naphthalene
degradation pathway genes reported from various Pseudomonas
genomes. The draft genome of CSV86 was aligned against
complete genome of P. stutzeri CCUG 29243 (NC_018028),
Pseudomonas sp. ND6 plasmid pND6-1 (NC_005244), P. putida
plasmid NAH7 (NC_007926) and P. fluorescens strain PC20
plasmid pNAH20 (AY887963).
Prediction of GIs and mobile genetic elementsTo predict the GIs, GC-profile, a web based tool [21] was used
to compute the GC content variation in DNA sequences. These
islands are marked by certain features such as the presence of
mobility genes, difference in the G+C content as compared to the
rest of genome, codon usage, tRNA genes and direct repeats [22].
Some of these features were manually identified in the genome for
the validation of GIs. Also, conserved insertion sequences (IS)
elements in CSV86 genome were identified using IS Finder
(http://www-is.biotoul.fr/) to further support the presence of GI
[23].
Validation of selected genotype by wet experimentsGrowth. Strain CSV86 was grown on 150 ml minimal salt
medium (MSM) [5] in 500 ml capacity baffled Erlenmeyer flasks
at 30uC on a rotary shaker (200 rpm) supplemented aseptically
with vanillin, veratraldehyde, ferulic acid, phenylalanine or
tyrosine (0.1%). The cell growth was observed spectrophotomet-
rically at 540 nm.
Preparation of cell-free extracts. CSV86 cells grown on
phenylalanine (0.1%) or glucose (0.25%) till late-log phase
were harvested and washed twice with Tris-malaete buffer
(200 mM, pH 6.0). Cells were re-suspended in ice-cold Tris-
malaete buffer (1:4 [wt/vol]) and sonicated at 4uC with four cycles
of 15 pulses each (1 s pulse, 1 s interval, cycle duration 30 s,
output 15 W) using an Ultrasonic processor (GE130). The cell
lysate was centrifuged at 37,000 6g for 30 min. The clear
supernatant obtained was referred to as the cell-free extract and
used as the source of enzyme. Protein was estimated by the
method of Bradford [24] using bovine serum albumin as the
standard.
Enzyme assay. Homogentisate 1,2-dioxygenase activity was
monitored by measuring the rate of O2 consumption at 30uC using
an oxygraph (Hansatech) fitted with a Clarke’s O2 electrode. The
reaction mixture (1 ml) contained Tris-malaete buffer (200 mM,
pH 6.0), homogentisate (2.5 mM) and an appropriate amount of
enzyme. The enzyme activity was calculated as nmol of O2
consumed per min. The specific activity is reported as nmol of O2
consumed min21 mg21 protein.
Heavy metal resistance. CSV86 was grown on 150 ml
modified minimal salt medium (MSM, medium contained Tris,
8 g; KH2PO4,0.2 g; NH4NO3, 1 g; MgSO4.7H2O, 100 mg;
MnSO4.H2O, 1 mg; CuSO4.5H2O, 1 mg; FeSO4.7H2O, 5 mg;
H3BO3, 1 mg; CaCl2.2H2O , 1 mg; NaMoO4, 1 mg; pH 7.5) in
500 ml capacity baffled Erlenmeyer flasks at 30uC on a rotary
shaker (200 rpm) supplemented aseptically with naphthalene
(0.1%) or glucose (0.25%) and appropriate concentration of heavy
metals such as copper, cadmium or cobalt (0.5 or 1 mM) and the
growth was monitored.
Results and Discussion
Pseudomonas putida CSV86 genome features andcomparative genomics
The 6,469,780 bp draft genome of CSV86 is almost close to
sequenced Pseudomonas genomes (Table S1); and assembled into
209 contigs that have been annotated by NCBI PGAAP into 5,836
coding sequences (CDSs) as shown in Table 1. RAST analysis
divided CSV86 genome into different metabolic subsystems
including catabolic pathways for various aromatic compounds
(Figure S1). The phylogenetic tree of 16S rRNA gene of CSV86
showed taxonomic relationship with Pseudomonas putida S16 sharing
98% homology (Figure 1). This was further supported by SIS
program, wherein CSV86 draft genome was assembled into 8
scaffolds (around 0.3 Mb of the genome was unmapped in the
scaffold) against P. putida S16. The analysis of ordered draft
genome of CSV86 with BRIG software showed ,70% identity
with P. putida S16, P. putida KT2440 and P. entomophila L48 except
P. stutzeri CCUG 29243 (,70%) (Figure 2); with gaps observed in
the region 6100–6500 kbp. Similarity search of the gapped region
using BLASTn with default parameters, revealed genes coding for
chromosome replication initiator protein dnaA and other proteins
involved in replication. A gene cluster with dnaA gene was
identified i.e. rnpA-rpmH-dnaA-dnaN-recF-gyrB. The oriC (replication
origin) has been reported to be present in this intergenic region
[25,26]. Therefore, it may be postulated that oriC region is located
in this region of CSV86 genome.
Genome Analysis of Pseudomonas putida CSV86
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Mining of aromatic compound degradation pathways inCSV86
The industrial revolution has led to the introduction of new
pollutants in the environment; which also ushered the evolution of
new catabolic pathways [27]. The absence or withdrawal of such
selective pressures often leads to the loss of the catabolic property,
if it is plasmid mediated; and even in cases of genome
organization, where it is associated with MGEs such as GIs.
These features play a significant role in the evolution of
community where these evolved microbes can be ideal candidates
for effective bioremediation either alone or in consortium [28].
The in-silico analysis of the CSV86 genome revealed the genes
coding for enzymes involved in the metabolic pathways which are
biochemically characterized earlier from CSV86 (Figure 3) and
their arrangement on the genome (Figure 4) as well as newly
Figure 1. Phylogenetic neighbor-joining tree of Pseudomonas putida CSV86. The tree is constructed from 16S rRNA gene sequences from 38completely sequenced Pseudomonas spp. The phylogenetic analysis was performed using MEGA 5.2 and the resultant Maximum Likelihood treeshows close taxonomic relationship of P. putida CSV86 to P. putida S16.doi:10.1371/journal.pone.0084000.g001
Table 1. Features associated with genome of P. putida CSV86according to NCBI PGAAP.
FEATURE CHROMOSOME
Length (bp) 6,469,780 bp
Number of contigs? 209
GC content (%) 61.85
Sequencing coverage 61.086
t-RNA genes 60
CDSs* 5,836
Hypothetical proteins 1,689
*CDSs: coding region, coding sequence.?There are 228 contigs according to Newbler Ver2.0, 454 assembly tool.doi:10.1371/journal.pone.0084000.t001
Genome Analysis of Pseudomonas putida CSV86
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identified pathways for the catabolism of aromatic compounds
(Figure 5).
Naphthalene degradation pathway. CSV86 can utilize
naphthalene and its derivates such as 1- and 2-methylnaphthalenes
as the sole source of carbon and energy via ring-hydroxylation
pathway (Figure 3), while side-chain hydroxylation pathway leads
to its detoxification [4,5]. In CSV86, naphthalene catabolic
pathway is initiated by naphthalene 1,2-dioxygenase (a three-
component system) which catalyzes the hydroxylation of the
aromatic ring to yield 1,2-dihydroxynaphthalene as a upper
pathway (contig 105). This diol is further sequentially oxidized to
catechol via lower pathway (contig 69), which enters the
tricarboxylic acid cycle (TCA) after meta ring-cleavage (Figure 3)
[4]. Using BLASTp, amino acid sequences of CSV86 naphthalene
degrading upper and lower pathway genes (Table S2) were
compared with that of other reported bacteria. It was observed
that upper pathway amino acid sequences shares higher homology
and hence are more conserved than that in lower pathway (Table
S3).
Both the nah and sal operon of CSV86 showed similarity with
Pseudomonas putida NCIB 9816-4 plasmid pDTG1 and Pseudomonas
sp. ND6 plasmid pND6-1 (Figure 4A & 4B; Figures S2 & S3).
Interestingly, in contig 105, the arrangement of genes observed
was tRNAGly, integrase followed by nah operon in the order
nahAa,Ab,Ac,Ad, BFCED. This arrangement is a characteristic
feature of a GI for e.g. clc element [29]. The sal operon consists of
9 genes organized as nahGTHILMOKJ with the regulatory gene
nahR present downstream of nahG gene. The regulation of nah
genes is controlled by nahR, which is in turn induced by salicylate
[30,31]. In CSV86 a transposase encoding gene is present
upstream of nahR gene which is missing in the plasmids being
compared (Figure 4B & Figure S3).
Regulation of nah operon. The genome sequence analysis
of CSV86 revealed that the naphthalene pathway is under the
control of LysR family of transcription regulators (LTTRs)
Figure 2. BLAST comparison of draft genome of Pseudomonas putidaCSV86 against four Pseudomonas species, using BRIG. Theinnermost rings depict GC content (Black) and GC Skew (purple/green) followed by concentric rings of query sequences colored according to BLASTidentity. The outermost rings depict genomes of the following microbes- P. putida S16 (Red), P. putida KT2440 (Pink) P. entomophila L48 (Blue), and P.stutzeri CCUG 29243 (Green).doi:10.1371/journal.pone.0084000.g002
Genome Analysis of Pseudomonas putida CSV86
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[32,33]. We have analyzed the differential regulation of naphtha-
lene degradation pathway in CSV86 with P. stutzeri CCUG 29243
(NC_018028) (chromosomally coded) and Pseudomonas putida
plasmid NAH7 DNA, strain G7 (AB237655). NahR protein is
essential for the activation of both the upper and lower operon of
naphthalene pathway in the presence of salicylate [34]. The
binding site for NahR with the promoter for Pnah and Psal are
reported to be located at 60 bp upstream to transcriptional start
site [34,35]. Therefore, the promoter data for nahAa (upper
pathway), nahG (lower pathway) and also the coding sequences for
NahR protein was analyzed. The consensus binding sequences of
NAH7 promoter has two cis-acting elements situated 6 bp apart
that interact with NahR protein [36]. The nahAa promoter of
CSV86 and P. stutzeri are identical with the reported NAH7
binding site for NahR protein. Both these promoters have an
additional cis-acting element with one base pair substitution and
4 bp spacing between the cis-acting elements. Whereas, nahG
promoter has a base pair substitution (TGAT is changed to
TAGT) in both the chromosomal promoters, with 4 bp separating
the two cis-acting elements (Table 2). A phylogenetic tree of nahR,
nahAa and nahG promoter sequences was also constructed. In all
the three promoter sequence comparisons, CSV86 and P. stutzeri
were grouped in same cluster (Figure S4).
The NahR of CSV86 interestingly showed 100% identity with
protein from P. stutzeri (chromosomally located) as compared to
81% identity with NAH7 (plasmid encoded) protein. The
substitution of methionine to isoleucine in NahR protein of
NAH7 altered the specificity of protein to salicylate and allowing
salicylate analog like benzoate to act as an inducer [37]. In CSV86
and P. stutzeri, NahR protein at 116th position has isoleucine
(Figure S5). However in CSV86, the enzymes responsible for
naphthalene and salicylate degradation are inducible in nature.
Benzoate does not induce these operons as the benzoate grown
cells failed to respire on naphthalene or salicylate and showed no
activity of enzymes involved in naphthalene or salicylate
degradation [5].
Benzoate degradation pathway. In CSV86 benzoate deg-
radation is initiated with the incorporation of molecular oxygen by
benzoate dioxygenase (encoded by benABC genes, a two-compo-
nent system) to yield catechol which enters the central carbon
metabolism via b-ketoadipate pathway after ortho-cleavage
(Figure 3) [5,38]. The details of the genes for benzoate degradation
that are present in contigs 175, 103, 118 and 116 are described in
Table S2. The CSV86 genome has the presence of complete
benzoate utilization system including the regulatory option of the
benABC operon i.e., transcriptional activator BenR with benzoate
Figure 3. The metabolic pathways for aromatic compounds in Pseudomonas putidaCSV86. Enzymes involved are: a, naphthalenedioxygenase; b, 1,2-dihydroxynaphthalene dioxygenase; c, salicylaldehyde dehydrogenase; d, salicylate hydroxylase; e, catechol 2,3-dioxygenase; f,catechol 1,2-dioxygenase; g, benzyl alcohol dehydrogenase; h, benzaldehyde dehydrogenase; i, 3,4-dihydroxybenzoate-3,4-dioxygenase; j, 4-hydroxyphenylacetic acid hydroxylase; k, 3,4-dihydroxyphenylacetic acid dioxygenase; l, homogentisate 1,2-dioxygenase; m, phenylacetyl-CoA ligase.Enzymes with wide-substrate specificity involved in various pathways in CSV86 are indicated in square bracket (4, 5, 6, 7).doi:10.1371/journal.pone.0084000.g003
Genome Analysis of Pseudomonas putida CSV86
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as effector; and utilization of catechol regulated via CatR with
cis,cis-muconate as effector [38]. In P. fluorescnes Pf0-1 the catBC
and catR genes are located between genes encoding benzoate MFS
transporter and catechol 1,2-dioxygenase while, in CSV86 these
genes are located upstream to the benzoate cluster. The catBC
genes are absent in this cluster of P. putida KT2440 (Figure 4C,
Figure S6).
The optional genes for benzoate degradation were also
identified in CSV86 genome, which encode p-hydroxybenzoyl-
CoA thioesterase (catalyses the conversion of p-hydroxybenzoyl-
CoA to p-hydroxybenzoate) and 5-carboxymethyl-2-hydroxymu-
conate Delta-isomerase (catalyses the conversion of 5-carboxy-
methyl-2-hydroxymuconate to form 5-carboxy-2-oxohept-3-ene-
dioate) located in contig 60 and 103, respectively. Besides salicylate
hydroxylase in contig 69 (sal operon), contig 103 also showed the
presence of an additional salicylate hydroxylase with 23% identity.
Aromatic alcohol degradation pathway. Although the
detoxification pathway of methylnaphthalenes closely resembles
to the side-chain hydroxylation of toluene degradation, CSV86
failed to utilize toluene or xylene as the sole source of carbon and
energy. Interestingly, strain could grow on benzyl alcohol, 2- and
4-hydroxy benzyl alcohol (Figure 3) [5]. The key enzymes of the
aromatic alcohol metabolism, benzyl alcohol dehydrogenase
(BADH) and benzaldehyde dehydrogenase (BZDH), have been
purified and were found to be wide-substrate specific and shown to
catalyze the conversion of 1- and 2-hydroxymethylnaphthalene to
respective naphthoic acids (dead end products) [5,6].
In CSV86, the gene cluster encoding BZDH and BADH was
located in contig 119. The proposed BZDH or NAD+-dependent
aryl aldehyde dehydrogenase gene was located adjacent to BADH,
aryl alcohol dehydrogenase gene in CSV86 and shares homology
with Pseudomonas putida DOT-T1E (aldehyde dehydrogenase,
87%). In CSV86 gene encoding for transcriptional regulator
(AraC family) was located downstream to the gene encoding
putative benzaldehyde dehydrogenase oxidoreductase protein,
which is absent in the same cluster of Pseudomonas putida GB-1
and Burkholderia sp. (Figure 4D, Figure S7, Table S2).
Phenylacetic and p- hydroxyphenylacetic acid
degradation pathway. CSV86 metabolizes phenylacetic acid
(PA) and p-hydroxyphenylacetic acids (HPA) using an unconven-
tional ‘aerobic hybrid’ pathway (Figure 3). PA is first activated to
its CoA-thioester (phenylacetyl CoA) by phenylacetyl-CoA ligase,
which through a series of CoA-thioester intermediates ultimately
leads to the formation of succinyl-CoA and acetyl-CoA that enters
the central metabolic pathway (Figure 3) [7,39]. In CSV86 the PA
catabolic genes (contig 88, Table S2) were organized in the order
as observed in Pseudomonas putida W619 and P. putida GB-1
(Figure 4E, Figure S8).
4-HPA degradation pathway in CSV86 follows homoprotoca-
techuate route which involves initial hydroxylation of 4-HPA
followed by ring cleavage (Figure 3) [7]. The pathway genes of
Figure 4. Gene organization of aromatic degradation pathways reported to be functionally characterized from Pseudomonas putidaCSV86. A. Naphthalene pathway (Contig 105), B. Salicylate pathway (Contig 69), C. Benzoate pathway (Contig 175), D. Aromatic alcohol degradationpathway (Contig 119), E. Phenylacetic acid pathway (Contig 88), F, Hydroxylphenylacetic acid pathway(Contig 7) and G–I, 4-Hydroxybenzoate(Contig 107, 99, 118). For details refer to Figures S2, S3, S6, S7, S8, S9, S10.doi:10.1371/journal.pone.0084000.g004
Genome Analysis of Pseudomonas putida CSV86
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4-HPA in CSV86 were present in contig 7, 120, 175 and 216
(Table S2). There were two copies of each hpaD (contig 7 and 120)
and homoprotocatechuate degradative operon repressor gene
(contig 7 and 216). The genes present in CSV86 contig 7 were
organized as hpaAGEDFHI while, in Pseudomonas fluorescens SBW25
homoprotocatechuate degradative operon repressor gene was
present upstream of this hpa cluster (Figure 4F, Figure S9).
p-Hydroxybenzoate degradation pathway. In CSV86, p-
hydroxybenzoate degradation is initiated by ring-hydroxylating 4-
hydroxybenzoate 3-monooxygenase (PobA) to yield 3,4-dihydrox-
ybenzoate (protocatechuate) which is further metabolized by ortho
ring-cleavage to yield carboxy-cis,cis-muconate by protocatechuate
dioxygenase (PcaGH, Figure 3). The later part of the pathway was
Figure 5. New aromatics degradation pathways genes identified in Pseudomonas putida CSV86 by genome analysis. A–B.Phenylpropanoid pathway genes (Contig 115, 220), C–E. Homogentisate pathway genes (Contig 27, 99, 177), F–H. Copper resistance genes (Contig19). For details refer to Figures S11, S12, S13.doi:10.1371/journal.pone.0084000.g005
Table 2. Consensus sequence of nahR binding site in nahAa and nahG gene obtained from P. putida plasmid NAH7.
Promoter NahR binding site
pNah7 -CGCAnTATTCAyGyTGuTGATnnAnnAnnTnnn-
PnahAa
CSV86 -GACAT TATTCATATTAGTGAT ACTAA TATTCA TTTATGGT TTATTGAC-
P. stutzeri -GACAT TATTCATATTAGTGAT ACTAA TATTCATTTATGGT TTATTGAC-
PnahG
CSV86 -TAGTG TATTTATCAATAGT TATGGCTTCGCTACTGTT-
P. stutzeri -TAGTG TATTTATCAATAGT TATGGCTTCGCTACTGTT-
Table shows consensus sequence of NahR binding site form P. putida plasmid NAH7 as reported by Schell et al., 1989. The sequences used in Figure S4, shown thehomologous cis-acting NahR regulated elements of nahAa and nahG genes (nah and sal operons, respectively) in case of P. putida CSV86 and P. stutzeri CCUG 29243genomes. The bold type face alphabets indicate nucleotides required for NahR activation of NahR-regulated promoters. (n: no nucleotide preference, Y: pyrimidine; U:purine).doi:10.1371/journal.pone.0084000.t002
Genome Analysis of Pseudomonas putida CSV86
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catalyzed by the pcaBCDIJF gene products which are also involved
in the degradation of benzoate.
The genes for 4-hydroxybenzoate pathway were distributed in
contig 99 (pcaHG), 107 (pobA), 118 (pcaRKFTBDC) and 175 (pcaIJ,
Table S2) in CSV86. Like Pseudomonas fluorescens Pf-5 and P. putida
GB-1, pobA gene in CSV86 (contig 107) was located downstream
to the gene encoding transcriptional regulator PobR (AraC family)
(Figure 4G, Figure S10A). The pcaHG genes were located in contig
99 and present downstream of zinc metalloprotease superfamily,
as observed in P. entomophila L48 and P. putida KT2440 (Figure 4H,
Figure S10B). A transporter, PcaK, is involved in the transport of
4-hydroxybenzoate across the membrane and reported to be
located in between pcaR and pcaF in P. putida, as can also be seen in
contig 118 in CSV86 (Figure 4I, Figure S10C). The expression of
pcaK gene has been shown to be repressed by benzoate, suggesting
cells prefer benzoate instead of 4-HPA when given together [40].
The pca genes have been shown to be arranged in a single cluster
in P. fluorescens [41], while in CSV86 they were segregated in
different contigs (Figure 4, Table S2, Figure S10).
Identification of additional aromatic compounddegradation pathways
Phenylpropanoid degradation pathway. Although lignin
degradation pathways are shown in fungi by enzymes such as
lignin peroxidases, manganese peroxidases, laccases etc [42];
aromatic degrading bacterial species are also reported to
metabolize lignin [43]. Vanillin, ferulic acid, veratraldehyde,
coniferyl aldehyde, b-coniferylether are identified as degradation
intermediates of lignin. In Pseudomonas sp. strain HR199, the
feruloyl-CoA synthetase (encoded by fcs) activates ferulic acid to its
CoA-thioester followed by hydration and non-oxidative cleavage
by enoyl-CoA hydratase/aldolase (encoded by ech) to form vanillin
and acetyl Co-A [44]. Vanillin is further converted to vanillate and
later protocatechuate by vanillin dehydrogenase (encoded by vdh)
and vanillin monooxygenase (encoded by vanAB) or vanillate-O-
demethylase (encoded by vanA and vanB), respectively [41,44]. In
CSV86, genes involved in the phenylpropanoid degradation were
segregated in different contigs (Figure 5A&B, Table S2, Figure
S11). The fcs-vdh-ech genes formed a cluster in contig 115
(Figure 5A, Figure S11A), vanAB genes in contig 119; and
Vanillate O-demethylase oxygenase subunit, vanA and vanillate
O-demethylase oxidoreductase, vanB along with the transcriptional
regulator for ferulate or vanillate catabolism in contig 220
(Figure 5B, Figure S11B).
Strain CSV86 showed good growth on MSM supplemented
with vanillin, veratraldehyde, or ferulic acid (0.1%). These
observations suggest that the lignin degradation intermediates
can be used as the sole source of carbon and energy and reflects
the existence of functional phenylpropanoid metabolic pathway in
CSV86. However, cells failed to utilize lignin (lignin sulphonic
acid) as the sole source of carbon and energy (data not shown).
Homogentisate degradation pathway. Homogentisate is a
metabolic intermediate of aromatic amino acid pathways.
Phenylalanine via tyrosine is converted to 4-hydroxyphenylpyr-
uvate (by PhhABC). The generated 4-hydroxyphenylpyruvate, is
then transformed to homogentisate (2,5-dihydroxyphenylacetic
acid) by 4-hydroxyphenylpyruvate dioxygenase (encoded by hpd
gene) [41]. Homogentisate 1,2-dioxygenase (HmgA) is the first
enzyme of the homogentisate pathway which catalyses the
transformation of homogentisate to 4-maleylacetoacetate. Isomer-
ization of 4-maleylacetoacetate by maleylacetoacetate isomerase
(HmgC) leads to the formation of fumarylacetoacetate, which is
finally hydrolyzed by fumarylacetoacetase (HmgB) generating
fumarate and acetoacetate [45,46]. The hpd gene is present along
with hmg genes in Pseudomonas syringae, Pseudomonas stutzeri and
Pseudomonas mendocina, whereas in P. putida these are scattered; in P.
aeruginosa the hpd gene is clustered with phh genes [41]. In CSV86,
phh, hpd and hmg genes were segregated in different clusters
(Figure 5C, D & E; Figure S12,Table S2). The phh genes were
present in the contig 177 with the arrangement of phhR
(phenylalanine hydroxylase transcriptional activator) and phhABC
genes similar to P. putida KT2440 and P. putida F1 (Figure 5C,
Figure S12C). The hpd gene was present in contig 99 (Figure 5D,
Figure S12B) and 134. The gene coding for transcriptional
regulator, TetR family, was present upstream to hpd gene in
CSV86 (contig 99), P. fluorescens Pf-5 and SBW25. The clustering
of hmg genes (contig 127) was similar to P. putida F1 and KT2440,
with the gene coding for transcriptional regulator (IclR family)
being transcribed in reverse direction to hmgABC genes (Figure 5E,
Figure S12A).
Strain CSV86 showed good growth on MSM supplemented
with phenylalanine or tyrosine (0.1%) as the sole source of carbon
and energy. Cell-free extract prepared from the cells grown on
phenylalanine showed the activity of homogentisate dioxygenase
(specific activity 49.9 nmol min21 mg21protein) while glucose
grown cells failed to do so. These results suggest that the
homogentisate pathway is functional in CSV86 and the enzyme
is inducible in nature (data not shown).
Identification of heavy metal resistance genes in CSV86Bioremediation of soils co-contaminated with heavy metals and
organics pose a major environmental challenge. Therefore,
bacteria harboring the properties of heavy metal resistance as
well as aromatic compound degradation would be highly
beneficial. The metal resistance is achieved by employing efflux
pumps or enzymatic detoxification or bioaccumulation (intracel-
lular/surface sequestration) or in combinations. Genes involved in
heavy metal resistance have been found to be encoded by plasmids
[47,48] or chromosome [49]. Chromosomal coded efflux system
for cadmium resistance has been reported in Bacillus as well as for
arsenic and antimony resistance in E. coli [50]. Cyanobacterium
synechocystis PCC6808 was also found to contain a homolog to Czc
(cadmium, zinc, and cobalt resistance system) and genes appar-
ently involved in arsenic and copper transport [51].
The genome of CSV86 was found to harbor heavy metal
resistance genes for copper (Figure 5F–H), cadmium, cobalt and
arsenic (Table S4). The copper resistance genes were dispersed in
CSV86 genome (contig 19, 153, 82) with a majority being in
contig 19 (Figure 5F, Figure S13A). The genes encoding copper
sensory histidine kinase (cusS), copper-sensing two-component
system response regulator (cusR) and copper tolerance were also
found to be located in the same contig (Figure 5G and H; Figure
S13B and C). Like copper, arsenic resistance genes were also
located in contig 153, with the exception of arsenic reductase gene
in contig 82. Genes for cobalt, chromate, cadmium, zinc and lead
resistance were also mapped during the genome analysis.
Strain CSV86 showed good growth on glucose or naphthalene
in the presence of heavy metals like copper, cadmium or cobalt at
0.5 as well as at 1 mM concentration, suggesting the ability of
strain to express the tolerance/resistance to these heavy metals
(data not shown).
Genetic bioaugmentationBioaugmentation using genetically engineered microorganisms
or consortia has been reported as an alternative strategy to
enhance the bioremediation of contaminated sites [52,53,54];
however the bio-safety issues pose a concern with these modified
bacteria. A wild isolate with its ability to transfer catabolic genes
Genome Analysis of Pseudomonas putida CSV86
PLOS ONE | www.plosone.org 8 January 2014 | Volume 9 | Issue 1 | e84000
through natural processes (HGT) such as conjugation, may
provide a better solution to contain and remediate these
compounds [55,56]. Pseudomonas putida IncP-9 TOL plasmid
pWW0, has been studied for genetic bioaugmentation of soil,
wastewater and aerobic microbial granules [57]. In another
example, dissemination of plasmid pGKT2 harboring catabolic
genes for hexahydro-1,3,5-trinitro-1,3,5,-triazine (RDX) degrada-
tion was studied by means of conjugation between the Gordonia sp.
KTR9 and the native population of the contaminated site, so as to
enhance the efficiency of bioremediation [58]. Genetic bioaug-
mentation via self-transmissible catabolic genes by donor bacteria
have stability issues in host bacteria. The other options for HGT
events are mediated by MGEs such as plasmids, GIs, transposons,
integrons and phages [55,56]. MGEs have been shown to play
significant role in supporting various types of genomic rearrange-
ment. HGT through GIs provides a better and desirable
advantage over plasmids as these elements are integrated in host
chromosome resulting in a stable genotype [3]. Therefore,
selective pressure for the survival of genotype is not essential for
better bioremediation capability and efficiency. These shuffling
introduce new gene clusters in a recipient bacteria guided through
stressed conditions of environment. IS elements are also associated
with transfer of metabolic loci and are therefore evolutionarily
significant in bacterial genomes. They are generally less than
2.5 kb in length and encode a protein that is involved in
transposition [59,60]. Using IS Finder, we report existence of
these elements in CSV86 genome (Table S5); however none of
these are present in vicinity to degradation pathway genes which
are associated with integrase (Figure 4A, Figure S2).
Attempts to isolate plasmid from CSV86 were unsuccessful.
Further, Southern hybridization suggests that naphthalene degra-
dation genes were localized in the genome. The naphthalene
degradation property of CSV86 can be transferred by conjugation
to Stenotrophomonas maltophila CSV89 with the transconjugants thus
obtained preferentially metabolizing aromatic compounds over
glucose. However, these properties were found to be unstable
when transconjugants were grown on rich medium [12]. These
results indicate probable involvement of GI in naphthalene
degradation capability of CSV86. Comparative analysis of
CSV86 genome with genome of established naphthalene degrad-
ing strains like P. stutzeri CCUG 29243, Pseudomonas sp. ND6
plasmid pND6-1, P. putida plasmid NAH7 and Pseudomonas
fluorescens strain PC20 plasmid pNAH20 was performed. Analysis
revealed that naphthalene and salicylate degrading gene clusters of
CSV86 and P. stutzeri CCUG 29243 shares a high degree of
homology at nucleotide sequence and showed the presence of
genes encoding integrase (Figure S14) and transposase (Figure S15)
upstream to both (nah and sal) operons. This feature was found to
be absent for naphthalene degrading genes encoded by plasmids
(pND6-1, NAH7 and pNAH20). This observation suggests the
presence of GI or conjugative element(s). GIs have specific
integration site (near or in tRNA gene) and lower G+C content
compared to rest of the genome. The GC-profile tool, which
calculates the compositional heterogeneity of DNA sequences, also
postulates the presence of GI in CSV86 genome. The analysis of
contig 105 which contained genes encoding for naphthalene upper
pathway revealed that there was a marked difference in G+C
content between the region containing naphthalene upper
pathway genes (90555–100230) with the rest of the contig DNA
(1–90554) suggesting possible insertion of GI in this region (Figure
S16). This is supported by the occurrence of genes encoding for
tRNA-Gly and integrase, located just upstream to the upper
pathway genes of naphthalene degradation. Neither tRNA-Gly
nor difference in the G+C content was observed in the contig 69
which encodes salicylate pathway.
Conclusions
The analysis of draft genome of Pseudomonas putida CSV86,
which encodes for 5836 ORFs revealed the presence of complete
catabolic pathway for naphthalene degradation with more than
95% homology with reported coding sequences for P. stutzeri
CCUG 29243. Identification of the GI at tRNAGly containing
naphthalene degradation genes supports the ability to transfer the
property by conjugation and the stability of this property. The
identification of additional degradative and metal resistance
genotype supported by phenotypic experiments further displays
the diversity of CSV86. The degradative capacities associated with
conjugation capabilities make this bacterium a possible donor in
safe dissemination of catabolic potential for the process of genetic
bioaugmentation.
Supporting Information
Figure S1 Subsystem distribution of Pseudomonas putida CSV86
genome in RAST.
(TIF)
Figure S2 Organisation and comparison of catabolic genes
involved in naphthalene degradation (nah operon) in P. putida
CSV86 (contig 105) against P. putida NCIB 9816-4 plasmid
pDTG1 (NC_004999), Pseudomonas sp. ND6 plasmid pND6-1
(NC_005244), Acidovorax sp. JS42 (NC_008782) and Leptothrix
cholodni SP-6 (NC_010524). Analysis was performed using RAST.
(TIF)
Figure S3 Organisation and comparison of catabolic genes
involved in salicylate acid degradation (sal operon) in P. putida
CSV86 (contig 69) against Pseudomonas sp. ND6 plasmid pND6-1
(NC_005244), P. putida NCIB 9816-4 plasmid pDTG1
(NC_004999), P. putida MT53 plasmid pWW53 (NC_008275)
and Dechloromonas aromatica RCB (NC_007298). Analysis was
performed using RAST.
(TIF)
Figure S4 Phylogenetic tree showing comparison of nahR (4A),
nahAa (4B) and nahG (4C) promoter sequences of P. putida CSV86
(AMWJ00000000), P.stutzeri CCUG 29243 (NC_018028) and P.
putida plasmid NAH7 (NC_007926).
(TIF)
Figure S5 Alignment of NahR amino acid sequence of P. putida
CSV86 (NZ_AMWJ01000062.1) with that of P. putida plasmid
NAH7 (NC_007926) and P.stutzeri CCUG 29243 (NC_018028).
The pink highlighted bar indicates residue 116: Methionine in
plasmid NAH7 and Isoleucine in both CSV86 and P. stutzeri.
Similarly, the blue highlighted bar indicates residue 248 (Arginine
in all the three); Green bar indicates residue 132 (Arginine in all
the three) and Grey bar indicates residue 169 (Asparagine in all the
three).
(TIF)
Figure S6 Organisation and comparison of catabolic genes
involved in the benzoate pathway in P. putida CSV86 (contig 175)
against P. fluorescens PfO-1 (NC_007492), P. putida KT2440
(NC_002947), P. putida F1 (NC_009512) and P. putida W619
(NC_010501). Analysis was performed using RAST.
(TIF)
Figure S7 Organisation and comparison of benzyl alcohol
dehydrogenase and benzaldehyde dehydrogenase genes in P.
Genome Analysis of Pseudomonas putida CSV86
PLOS ONE | www.plosone.org 9 January 2014 | Volume 9 | Issue 1 | e84000
putida CSV86 (contig 119) against P. putida GB-1 (NC_010322),
Burkholderia cenocepacia HI2424 (NC_008544), Burkholderia cenocepacia
AU 1054 (NC_008060) and Burkholderia ambifaria AMMD
(NC_008392). Analysis was performed using RAST.
(TIF)
Figure S8 Organisation and comparison of catabolic genes
involved in phenylacetic acid degradation in P. putida CSV86
(contig 88) against P. putida W619 (NC_010501), P. putida GB-1
(NC_010322), P. putida KT2440 (NC_002947) and P. putida F1
(NC_009512). Analysis was performed using RAST.
(TIF)
Figure S9 Organisation and comparison of catabolic genes
involved in 4-hydroxy phenylacetic acid degradation in P. putida
CSV86 (contig 7) against P. fluorescens SBW25 (NC_012660), P.
aeruginosa PA7 (NC_009656), P. aeruginosa UCBPP-PA14
(NC_008463) and P. aeruginosa PAO1 (NC_002516). Analysis was
performed using RAST.
(TIF)
Figure S10 Organisation and comparison of catabolic genes
involved in phydroxy benzoate degradation in P. putida CSV86
against P. fluorescens Pf-5 (NC_004129), P. putida GB-1
(NC_010322), P. putida W619 (NC_010501), P. putida KT2440
(NC_002947), P. entomophila L48 (NC_008027) , P. putida F1
(NC_009512), P. fluorescens SBW25 (NC_012660), P. syringae pv.
syringae B728a (NC_007005) and P .syringae pv. phaseolicola 1448A
(NC_005773). Analysis was performed using RAST.
(TIF)
Figure S11 Organisation and comparison of catabolic genes
involved in phenylpropanoid pathway in P. putida CSV86 against
P. putida F1 (NC_009512), P. putida W619 (NC_010501), P. putida
KT2440 (NC_002947), P. syringae pv. tomato str. DC3000
(NC_004578) and P. putida GB-1 (NC_010322). Analysis was
performed using RAST.
(TIF)
Figure S12 Organization and comparison of catabolic genes
involved in homogentisate pathway in CSV86 against P. putida
KT2440 (NC_002947), P. putida F1 (NC_009512), P. fluorescens Pf-
5 (NC_004129), P. putida W619 (NC_010501), P. fluorescens SBW25
(NC_012660), P. fluorescens PfO-1 (NC_007492), P. putida GB-1
(NC_010322) and P. entomophila L48 (NC_008027). Analysis was
performed using RAST.
(TIF)
Figure S13 Organisation and comparison of catabolic genes
involved in copper resistance in P. putida CSV86 against P. putida
GB-1 (NC_010322), P. putida W619 (NC_010501), P. entomophila
L48 (NC_008027), P. putida KT2440 (NC_002947), P. putida F1
(NC_009512), P. mendocina ymp (NC_009439) and P. stutzeri A1501
(NC_009434). Analysis was performed using RAST.
(TIF)
Figure S14 Progressive alignment between the draft genomes of
P. putida CSV86 and the complete genome of P. stutzeri CCUG
29243 (NC_018028), Pseudomonas sp. ND6 plasmid pND6-1,
complete sequence (NC_005244) and P. putida plasmid
NAH7(NC_007926) and P. fluorescens strain PC20 plasmid
pNAH20 (AY887963). Naphthalene degrading upper operon has
been aligned with all the 4 genomes. Integrase coding genes has
been highlighted by the black vertical bar. Colored blocks outline
genome sequence that align to part of another genome, and is
presumably homologous and internally free from genomic
rearrangement (Locally Colinear Blocks or LCBs). The white
colored area indicates regions with no alignment as they may
probably contain sequences specific to that genome. The inverted
blocks below the centre line indicate regions that align in the
reverse complement (inverse) orientation and the height of the
colored bars signify the nucleotide sequence similarity.
(TIF)
Figure S15 Progressive alignment between the draft genomes of
P. putida CSV86 and the complete genome of P. stutzeri CCUG
29243 (NC_018028), Pseudomonas sp. ND6 plasmid pND6-1
(NC_005244) and Pseudomonas putida plasmid NAH7(NC_007926)
and P. fluorescens strain PC20 plasmid pNAH20 (AY887963) using
MAUVE software. Naphthalene degrading lower operon has been
aligned with all the 4 genomes. Transposase coding genes has been
highlighted by the black vertical bar.
(TIF)
Figure S16 Output image of GC Profile software using
nucleotide sequence of contig 105 having naphthalene degrada-
tion upper pathway gene. The arrow indicates the segmentation
point 1 from where the GC content varies as compared to rest of
the contig sequence. The GC content of 1–90554 bp region is
63.8, whereas the region after the segmentation point i.e. 90555–
100230 bp has GC content of 51.64 (segmentation strength-
258.32).
(TIF)
Table S1 Summary of P. putida CSV86 draft genome compared
with other complete genome of Pseudomonas spp. available in
KEGG database.
(DOC)
Table S2 Pathways present in P. putida CSV86 genome based on
NCBI PGAAP annotation.
(DOCX)
Table S3 Percentage homology of Naphthalene upper and
lower operon pathway proteins in P. putida CSV86 with
homologous proteins of 4 Pseudomonas species reported for
naphthalene degradation capability.
(DOCX)
Table S4 Heavy metal resistance genes identified in P. putida
CSV86 genome annotation in RAST and their percentage
homology with the closest respective gene.
(DOCX)
Table S5 Mobile genetic elements present in P. putida CSV86
genome located using IS finder.
(DOCX)
Author Contributions
Conceived and designed the experiments: HJP PSP. Performed the
experiments: VP SCR AM. Analyzed the data: VP SCR AM PSP HJP.
Contributed reagents/materials/analysis tools: HJP PSP. Wrote the paper:
VP HJP PSP SCR AM.
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